The present invention relates to the prevention and remediation of biofilms in dental equipment.
Over the years the dental services community has become aware that the water systems currently designed for general dental practice do not deliver water of an optimal microbiological quality. It has been documented that dental unit waterlines harbor a wide variety of microorganisms including bacteria, fungi, and protozoans, which adhere to the interior surfaces of the waterline tubing to form colonies.
The American Dental Association (ADA) has recommended an ambitious and aggressive effort to encourage industry and independent researchers to improve the design of dental equipment by the year 2000 so that water delivered to patients during nonsurgical dental procedures contains no more than 200 colony forming units (cfu) of aerobic mesophilic heteretrophic bacteria per milliliter at any time in the unfiltered output of the dental unit (equivalent to an existing quality assurance standard for dialysate fluid in hemodyalisis units). This represents a massive decrease in microbial contamination of dental unit water from values that frequently run over 10,000 cfu/mL under current practice.
The organisms that currently contaminate dental units originate from a variety of sources, but the water lines of dental units represent the largest single source of biofilms. These biofilms live on the luminal walls of the water lines within the dental unit itself. The presence of adherent microbial biofilms in dental unit water lines was first reported more than 30 years ago. Interest in this issue has recently escalated throughout the world because many studies have confirmed the magnitude and widespread occurrence of contamination. In fact, one study found that the water coming out of dental unit water lines was of uniformly poorer quality than water coming out of taps in the same rooms.
The predominant organisms in dental water lines are Pseudomonas and Legionella species. Pseudomnonas are the most common organisms, but Legionella represent perhaps the most dangerous of the organisms routinely found in dental unit water lines (DUWLs). In one recent study, Legionella organisms were found in 29 out of 47 dental units tested. In many cases the organisms were present at a concentration of  greater than 103 per 10 mL sample.
The fact that aerosols generated from water within dental operatories are the source responsible for the elevated seropositivity to Legionella antibodies for dental personnel has been confirmed by several research studies. An Austrian serological study analyzing samples from 107 dentists, dental assistants and technicians found that thirty four percent (34%) tested positive to the polyvalent L. pneumophila antigen (the species considered most pathogenic to humans) comparing to only five percent (5%) from a non-medical workers control group. The highest prevalence (50%) was demonstrated among dentists, followed by assistants (38%) and technicians (20%). In an analogous study in the United States, 20% of the students and employees at a dental clinic in Virginia were seropositive for Legionella antibodies. Even though the higher seroprevalence rates have not been directly correlated with higher rates of disease among dental personnel, investigators speculate that it may reflect continuous exposure to small numbers of organisms resulting in mild (Pontiac fever) or inapparent infections.
Microbiologists have traditionally focused on free-floating bacteria growing in laboratory cultures. Recently they have realized that 99% of bacterial activity in open ecosystems occurs in biofilms adhered to surfaces. By 1990, researchers confirmed that biofilm bacteria are morphologically and metabolically distinct from free-floating ones, and that any bacterium can form a biofilm, once it finds a place to stick. As a result, biofilms, which were once considered odd curiosities, today are one of the hottest topics in microbiology since their occurrence has consequences for everything from medical technology to oil recovery.
Any solid surface immersed in an aquatic environment immediately serves as an adhesion site for macromolecules and other, low-molecular-weight hydrophobic molecules present in the water. This forms a so-called conditioning film which alters surface characteristics (such as hydrophobicity) and enhances the efficiency of subsequent bacterial adhesion.
The fundamental process of biofilm formation may be either passive or active. Some microorganisms already possess the necessary tools, such as extracellular polymeric substances or fimbriae and can immediately passively attach to a surface. Other bacteria require prolonged exposure to the surface to attach firmly. The active biofilm formation is a time-dependent process that begins with an initial reversible association between the microbe and the surface. During this period a genetic cascade is set off that turns on specific genes to make polysaccharides.
Due to the secretion of these substances and subsequent microbial multiplication, an irreversible adhesion and colonization of the surface is achieved. The production of a continuous fixed biofilm on the surface is then a function of cell division within the described polymeric matrix and can include the physical inclusion of other bacteria, fungi and protozoa from the free-floating microbial community of the surrounding water. All this eventually creates a slime layer composed of columns permeated by water-filled spaces through which materials and microbial by-products flow. The attached microbes have several survival advantages, in comparison to free-floating microorganisms.
Dental water lines, with their high surface-to-volume ratio, intermittent pattern of operation (with short periods of flow alternating with long, stagnant periods), and the characteristic of fluid dynamics in narrow, smooth-walled channels with only laminar flow, provide an ideal environment for microbial colonization.
The recognition of this health threat comes at the same time as an increasing awareness of potential occupational hazards in the dental office and concerns about increasing numbers of immunocompromised patients, such as elderly people, people with AIDS, cancer patients, diabetics, persons with chronic organic disorders or autoimmune diseases, and people who have received organ transplants or have recently received blood transfusions, all of whom have diminished resistance to opportunistic pathogens. All of these factors provide motivation for improving the quality of water for dental procedures.
Current methods for reducing water contamination are based on purging water lines for an extended period daily and a short period between patients or using independent water systems. The former method requires diligently following the procedure, and only reduces the bacterial count temporarily, and even then the reduction is not enough to meet the new standards. The latter method is only effective until the water system is contaminated once, and is then no better than using tap water.
Semiautomatic chemical treatments have been introduced that can be effective, but they require regular attention to keep the system filled with the disinfecting agent, and require an inventory of the agent be kept on hand at all times.
The current recommendations from the CDC for reducing the risk of contamination from dental unit water lines (DUWLs) involve a series of steps that must be followed conscientiously if they are to succeed. The first of these is the installation and use of anti-retraction valves (check valves to limit flow in a line to one direction) on all water and air lines. These devices prevent saliva and other fluids from the patient from being sucked back into water lines and colonizing the biofilm present in the water line. This is a concern because saliva from a patient""s mouth is more likely to contain pathogenic organisms than the incoming water from a municipal water system. Accordingly, these valves must be regularly maintained to remain effective.
Pasteurization is widely used in the food industry, particularly for dairy products, but less often for drinking water, where chemical disinfection is usually preferred. Water pasteurization has received attention in recent years in two arenas. Solar heated pasteurization is generally viewed as a viable approach to improving the quality of drinking water in undeveloped areas. Pasteurization has been adapted by Murikami, et. al., to eliminate microorganisms from water in an ultra-pure water system.
A variety of values have been cited in the published literature for the times and temperatures required to kill waterbome microbes. Andreatta et. al., designed their system to reach 65xc2x0 C. for at least a few minutes. A Mexican study concluded that 68xc2x0 C. was the preferred pasteurization temperature, but this difference is insignificant. Filtration, to remove multicellular organisms, combined with two hours at 70xc2x0 C. were recommended for sterilizing natural sea water. Goldstein demonstrated that a two minute exposure to a temperature of 76xc2x0 C. was sufficient to kill up to 99.8% of all microorganisms in contaminated well water. Since the time needed to kill most organisms declines exponentially with increasing temperature, a 20xc2x0 C. increase in treatment temperature produces an approximately four-fold reduction in time, and a 30xc2x0 C. increase in temperature produces an approximately eight-fold reduction in time. In related work, Charm used a temperature of 75xc2x0 C. for 0.05 seconds to kill viruses in blood.
The effectiveness of hydrogen peroxide as a general-purpose disinfectant is well recognized and its antibacterial effects have been studied and reviewed by a number of authors in recent years. Hydrogen peroxide can be synthesized electrochemically by the reduction of oxygen at the cathode of an electrochemical cell. The reduction of oxygen in both acidic and basic media has been extensively reported in the literature. In basic media oxygen reduction occurs either in a single four electron reduction to produce hydroxyl ions (shown in equation 1),
O2+2H2O+4exe2x88x92xe2x86x924OHxe2x88x92xe2x80x83xe2x80x83Eq. 1
or in two discrete steps, (equations 2 and 3),
O2+2H2O+2exe2x88x92xe2x86x92H2O2+2OHxe2x88x92xe2x80x83xe2x80x83Eq. 2
H2O2+2exe2x88x92xe2x86x922OHxe2x88x92xe2x80x83xe2x80x83Eq. 3
where the hydrogen peroxide is a stable reaction product, which can be utilized if it is promptly removed from the vicinity of the electrode to prevent further reduction. The mechanism of the reaction depends strongly on the type of cathode. Hydrogen peroxide is believed to be formed on iron during the rusting process. Carbon is reported to catalyze the hydrogen peroxide reaction in alkaline media while catalytic materials such as cobalt tetrakis(4-methoxyphenyl)porphyrin (CoTMPP) have been used to produce hydrogen peroxide under acidic conditions. Yields of 40% have been achieved with some carbon-supported porphyrin complexes.
Hydrogen peroxide, generated at the point of use, has already been demonstrated to offer particular advantages to the paper and pulp industries as a bleaching agent used to replace chlorine. The electrochemical method of production is cost effective for small scale operations. In the synthesis of hydrogen peroxide it has been suggested that a high surface area cathode is required for successful synthesis and the electrolyte must be free of any transition metal impurities as these catalyze the decomposition reaction to water.
The electrochemical reaction for the generation of hydrogen peroxide using a half fuel cell approach, based on proton exchange membrane (PEM) fuel cell technology is shown in Equations 4-6. On the anode side, humidified hydrogen gas is supplied to a porous electrocatalyst that breaks the hydrogen down to protons and electrons by the following reaction:
H2xe2x86x922H++2exe2x88x92xe2x80x83xe2x80x83Eq. 4
The hydrated protons diffuse through the membrane electroosmotically until they reach the cathode. The cathode material is an electrocatalyst to catalyze the two electron reduction of oxygen to peroxide, as shown in equation 5, which is formed as a surface species on the catalyst. During the cathodic reaction
O2+2exe2x88x92xe2x86x92O2xe2x88x92xe2x80x83xe2x80x83Eq. 5
the protons traversing the membrane react with the reduced oxygen species to form hydrogen peroxide, as shown in equation 6:
O2xe2x88x92+2H+xe2x86x92H2O2xe2x80x83xe2x80x83Eq. 6
The hydrogen peroxide is then transported from the cell by the water present in the cell.
Thomas et. al., report that a concentration of at least 0.1% H2O2 is required for broad bactericidal action over a short time span (minutes), with higher concentrations (on the order of 1 to 3%) being effective in seconds, and substantially lower concentrations (as low as 0.0007%) suppressing bacterial growth (bacteriostatic action). Juven and Pierson report a similar concentration for bacteriostatic activity, 0.0005%, with 0.0034% marking the onset of bactericidal activity and 3% being quite effective against a broad range of pathogens. Lever and Sutton report effective bactericidal action in less than an hour for concentrations of 0.05% to 0.10%, increasing concentrations reducing the time required. These levels were also useful against viruses. The lowest concentration of hydrogen peroxide described as effective as a germicide comes from commercial formulations for hard surface cleaning agents. According to 21 CFR Part 178.1010 (c) (39), a concentration of 72 ppm hydrogen peroxide is the minimum level for use in an acidic sanitizing solution for use on food utensils and food handling surfaces.
Under current FDA rules, aqueous solutions of hydrogen peroxide with hydrogen peroxide concentrations between 1.5 and 3.0% are generally recognized as safe and effective for use as wound cleansing and healing agents for use in the mouth, and may be sold as such xe2x80x9cover the counterxe2x80x9d without restriction.
There are a number of products currently on the market that fall under this FDA rule. For example, two over-the-counter products include Colgate Peroxyl(copyright) Hygienic Dental Rinse (containing 1.5% hydrogen peroxide) and Metadent(copyright) toothpaste.
Most isolated, or clean, water systems cannot be sterilized. With these systems the only protection the system has is the diligence of the practitioner in only using sterile water and in following the manufacturer""s instructions for periodic disinfection.
Besides being overly reliant on the diligence of the practitioner, current systems may also rely on an inventory of materials, such as a solution of hydrogen peroxide, to maintain their effectiveness. A diligent practitioner can maintain an inventory of any chemicals required, and use them regularly, but if the chemicals degrade in storage, as hydrogen peroxide does, all of this effort is for naught. Using under strength reagents is worse than no treatment program at all, since the act of using the weak materials will give the practitioner a false sense of security that they would not have in the absence of a treatment program.
Consequently, there is a demonstrated need for a system to mitigate the effects of biofilms by preventing the formation of biofilms in clean dental unit water lines (DUWLs) and eliminating biofilms from existing DUWLs. It would be desirable if the system did not heavily rely upon diligence of a practitioner or require reagents to maintain water quality.
The present invention provides a system for the prevention and remediation of biofilms in water lines, such as dental unit water lines. The system requires only electricity and the water being used by the dental unit to operate. The system consists of a series of steps, or unit operations, which together prevent the initial formation of a biofilm by preventing the entrance of microbes into the system and eliminates existing biofilms by introducing a disinfecting agent, hydrogen peroxide, capable of killing the organisms in the biofilm into the water stream. Any organisms not eliminated in these steps and much of the remains of the dead organisms are removed from the water stream by a final filter before the water is delivered to the patient. The action of this system can substantially reduce the risk of infection from water borne pathogens for both patients and practitioners.